Cancer diagnostic method based upon DNA methylation differences

ABSTRACT

There is disclosed a cancer diagnostic method based upon DNA methylation differences at specific CpG sites. Specifically, the inventive method provides for a bisulfite treatment of DNA, followed by methylation-sensitive single nucleotide primer extension (Ms-SNuPE), for determination of strand-specific methylation status at cytosine residues.

This patent application is a continuation of pending U.S. patentapplication Ser. No. 09/887,941 filed 22 Jun. 2001, which is acontinuation of U.S. patent application Ser. No. 09/094,207, filed 09Jun. 1998, now U.S. Pat. No. 6,251,594, which claims priority from U.S.Provisional Patent Application 60/049,231 filed 09 Jun. 1997.

TECHNICAL FIELD OF THE INVENTION

The present invention provides a cancer diagnostic method based upon DNAmethylation differences at specific CpG sites. Specifically, theinventive method provides for a bisulfite treatment of DNA, followed bymethylation-sensitive single nucleotide primer extension (Ms-SNuPE), fordetermination of strand-specific methylation status at cytosineresidues.

BACKGROUND OF THE INVENTION

Cancer treatments, in general, have a higher rate of success if thecancer is diagnosed early and treatment is started earlier in thedisease process. The relationship between improved prognosis and stageof disease at diagnosis hold across all forms of cancer for the mostpart. Therefore, there is an important need to develop early assays ofgeneral tumorigenesis through marker assays that measure generaltumorigenesis without regard to the tissue source or cell type that isthe source of a primary tumor. Moreover, there is a need to addressdistinct genetic alteration patterns that can serve as a platformassociated with general tumorigenesis for early detection and prognosticmonitoring of many forms of cancer.

Importance of DNA Methylation

DNA methylation is a mechanism for changing the base sequence of DNAwithout altering its coding function. DNA methylation is a heritable,reversible and epigenetic change. Yet, DNA methylation has the potentialto alter gene expression, which has profound developmental and geneticconsequences. The methylation reaction involves flipping a targetcytosine out of an intact double helix to allow the transfer of a methylgroup from S-adenosylmethionine in a cleft of the enzyme DNA(cystosine-5)-methyltransferase (Klimasauskas et al., Cell 76:357-369,1994) to form 5-methylcytosine (5-mCyt). This enzymatic conversion isthe only epigenetic modification of DNA known to exist in vertebratesand is essential for normal embryonic development (Bird, Cell 70:5-8,1992; Laird and Jaenisch, Human Mol. Genet. 3:1487-1495, 1994; andBestor and Jaenisch, Cell 69:915-926, 1992). The presence of 5-mCyt atCpG dinucleotides has resulted in a 5-fold depletion of this sequence inthe genome during vertebrate evolution, presumably due to spontaneousdeamination of 5-mCyt to T (Schoreret et al., Proc. Natl. Acad. Sci. USA89:957-961, 1992). Those areas of the genome that do not show suchsuppression are referred to as “CpG islands” (Bird, Nature 321:209-213,1986; and Gardiner-Garden et al., J. Mol. Biol. 196:261-282, 1987).These CpG island regions comprise about 1% of vertebrate genomes andalso account for about 15% of the total number of CpG dinucleotides(Bird, Infra.). CpG islands are typically between 0.2 to about 1 kb inlength and are located upstream of many housekeeping and tissue-specificgenes, but may also extend into gene coding regions. Therefore, it isthe methylation of cytosine residues within CpG islands in somatictissues, which is believed to affect gene function by alteringtranscription (Cedar, Cell 53:3-4, 1988).

Methylation of cytosine residues contained within CpG islands of certaingenes has been inversely correlated with gene activity. This could leadto decreased gene expression by a variety of mechanisms including, forexample, disruption of local chromatin structure, inhibition oftranscription factor-DNA binding, or by recruitment of proteins whichinteract specifically with methylated sequences indirectly preventingtranscription factor binding. In other words, there are several theoriesas to how methylation affects mRNA transcription and gene expression,but the exact mechanism of action is not well understood. Some studieshave demonstrated an inverse correlation between methylation of CpGislands and gene expression, however, most CpG islands on autosomalgenes remain unmethylated in the germline and methylation of theseislands is usually independent of gene expression. Tissue-specific genesare usually unmethylated and the receptive target organs but aremethylated in the germline and in non-expressing adult tissues. CpGislands of constitutively-expressed housekeeping genes are normallyunmethylated in the germline and in somatic tissues.

Abnormal methylation of CpG islands associated with tumor suppressorgenes may also cause decreased gene expression. Increased methylation ofsuch regions may lead to progressive reduction of normal gene expressionresulting in the selection of a population of cells having a selectivegrowth advantage (i.e., a malignancy).

It is considered that altered DNA methylation patterns, particularlymethylation of cytosine residues, cause genome instability and aremutagenic. This, presumably, has led to an 80% suppression of a CpGmethyl acceptor site in eukaryotic organisms, which methylate theirgenomes. Cytosine methylation further contributes to generation ofpolymorphism and germ-line mutations and to transition mutations thatinactivate tumor-suppressor genes (Jones, Cancer Res. 56:2463-2467,1996). Methylation is also required for embryonic development of mammals(Bestor and Jaenisch, Cell 69:915-926, 1992). It appears that that themethylation of CpG-rich promoter regions may be blocking transcriptionalactivity. Therefore, there is a probability that alterations ofmethylation are an important epigenetic criteria and can play a role incarcinogenesis in general due to its function of regulating geneexpression. Ushijima et al. (Proc. Natl. Acad. Sci. USA 94:2284-2289,1997) characterized and cloned DNA fragments that show methylationchanges during murine hepatocarcinogenesis. Data from a group of studiesof altered methylation sites in cancer cells show that it is not simplythe overall levels of DNA methylation that are altered in cancer, butchanges in the distribution of methyl groups.

These studies suggest that methylation, at CpG-rich sequences known asCpG islands, provide an alternative pathway for the inactivation oftumor suppressors, despite the fact that the supporting studies haveanalyzed only a few restriction enzyme sites without much knowledge asto their relevance to gene control. These reports suggest thatmethylation of CpG oligonucleotides in the promoters of tumor suppressorgenes can lead to their inactivation. Other studies provide data thatsuggest that alterations in the normal methylation process areassociated with genomic instability (Lengauer et al. Proc. Natl. Acad.Sci. USA 94:2545-2550, 1997). Such abnormal epigenetic changes may befound in many types of cancer and can, therefore, serve as potentialmarkets for oncogenic transformation, provided that there is a reliablemeans for rapidly determining such epigenetic changes. The presentinvention was made to provide such a universal means for determiningabnormal epigenetic changes and address this need in the art.

Methods to Determine DNA Methylation

There is a variety of genome scanning methods that have been used toidentify altered methylation sites in cancer cells. For example, onemethod involves restriction landmark genomic scanning (Kawai et al.,Mol. Cell. Biol. 14:7421-7427, 1994), and another example involvesmethylation-sensitive arbitrarily primed PCR (Gonzalgo et al., CancerRes. 57:594-599, 1997). Changes in methylation patterns at specific CpGsites have been monitored by digestion of genomic DNA withmethylation-sensitive restriction enzymes followed by Southern analysisof the regions of interest (digestion-Southern method). Thedigestion-Southern method is a straightforward method but it hasinherent disadvantages in that it requires a large amount of DNA (atleast or greater than 5 μg) and has a limited scope for analysis of CpGsites (as determined by the presence of recognition sites formethylation-sensitive restriction enzymes). Another method for analyzingchanges in methylation patterns involves a PCR-based process thatinvolves digestion of genomic DNA with methylation-sensitive restrictionenzymes prior to PCR amplification (Singer-Sam et al., Nucl. Acids Res.18:687,1990). However, this method has not been shown effective becauseof a high degree of false positive signals (methylation present) due toinefficient enzyme digestion of overamplification in a subsequent PCRreaction.

Genomic sequencing has been simplified for analysis of DNA methylationpatterns and 5-methylcytosine distribution by using bisulfite treatment(Frommer et al., Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992).Bisulfite treatment of DNA distinguishes methylated from unmethylatedcytosines, but original bisulfite genomic sequencing requireslarge-scale sequencing of multiple plasmid clones to determine overallmethylation patterns, which prevents this technique from beingcommercially useful for determining methylation patterns in any type ofa routine diagnostic assay.

In addition, other techniques have been reported which utilize bisulfitetreatment of DNA as a starting point for methylation analysis. Theseinclude methylation-specific PCR (MSP) (Herman et al. Proc. Natl. AcadSci. USA 93:9821-9826, 1992); and restriction enzyme digestion of PCRproducts amplified from bisulfite-converted DNA (Sadri and Hornsby,Nucl. Acids Res. 24:5058-5059, 1996; and Xiong and Laird, Nucl. AcidsRes. 25:2532-2534, 1997).

PCR techniques have been developed for detection of gene mutations(Kuppuswamy et al., Proc. Natl. Acad. Sci. USA 88:1143-1147, 1991) andquantitation of allelic-specific expression (Szabo and Mann, Genes Dev.9:3097-3108, 1995; and Singer-Sam et al., PCR Methods Appl. 1:160-163,1992). Such techniques use internal primers, which anneal to aPCR-generated template and terminate immediately 5′ of the singlenucleotide to be assayed. However an allelic-specific expressiontechnique has not been tried within the context of assaying for DNAmethylation patterns.

Therefore, there is a need in the art to develop improved diagnosticassays for early detection of cancer using reliable and reproduciblemethods for determining DNA methylation patterns that can be performedusing familiar procedures suitable for widespread use. This inventionwas made to address the foregoing need.

SUMMARY OF THE INVENTION

The present invention provides a method for determining DNA methylationpatterns at cytosine sites, comprising the steps of:

(a) obtaining genomic DNA from a DNA sample to be assayed;

(b) reacting the genomic DNA with sodium bisulfite to convertunmethylated cytosine residues to uracil residues while leaving any5-methylcytosine residues unchanged to provide primers specific for thebisulfite-converted genomic sample for top strand or bottom strandmethylation analysis;

(c) performing a PCR amplification procedure using the top strand orbottom strand specific primers;

(d) isolating the PCR amplification products;

(e) performing a primer extension reaction using Ms-SNuPE primers,[³²P]dNTPs and Taq polymerase, wherein the Ms-SNuPE primers comprisefrom about a 15 mer to about a 22 mer length primer that terminatesimmediately 5′ of a single nucleotide to be assayed; and

(f) determining the relative amount of methylation at CpG sites bymeasuring the incorporation of different ³²P-labeled dNTPs.

Preferably, the [³²P]NTP for top strand analysis is [³²P]dCTP or[³²P]TTP. Preferably, the [³²P]NTP for bottom strand analysis is[³²P]dATP or [³²P]dGTP. Preferably, the isolation step of the PCRproducts uses an electrophoresis technique. Most preferably, theelectrophoresis technique uses an agarose gel. Preferably, the Ms-SNuPEprimer sequence comprises a sequence of at least fifteen but no morethan twenty five, bases having a sequence selected from the groupconsisting of GaL1 (SEQ ID NO:1), GaL2 (SEQ ID NO:2), GaL4 (SEQ IDNO:3), HuN1 (SEQ ID NO:4), HuN2 (SEQ ID NO:5), HuN3 (SEQ ID NO:6), HuN4(SEQ ID NQ:7), HuN5 (SEQ ID NO:8), HuN6 (SEQ ID NO:9), CaS1 (SEQ IDNO:10), CaS2 (SEQ ID NO:11), CaS4 (SEQ ID NO:12), and combinationsthereof.

The present invention further provides a Ms-SNuPE primer sequencedesigned to anneal to and terminate immediately 5′ of a desired cytasinecodon in the CpG target site and that is located 5′ upstream from a CpGisland and are frequently hypermethylated in promoter regions of somaticgenes in malignant tissue. Preferably, the Ms-SNuPE primer sequencecomprises a sequence of at least fifteen bases having a sequenceselected from the group consisting of GaL1 (SEQ ID NO:1), GaL2 (SEQ IDNO:2), GaL4 (SEQ ID NO:3), HuN1 (SEQ ID NO:4), HuN2 (SEQ ID NO:5), HuN3(SEQ ID NO:6), HuN4 (SEQ ID NO:7), HuN5 (SEQ ID NO:8), HuN6 (SEQ IDNO:9), CaS1 (SEQ ID NO:10), CaS2 (SEQ ID NO:11), CaS4 (SEQ ID NO:12),and combinations thereof. The present invention further provides amethod for obtaining a Ms-SNuPE primer sequence, comprising finding ahypermethylated CpG island in a somatic gene am a malignant tissue orcell culture, determining the sequence located immediately 5′ upstreamfrom the hypermethylated CpG island, and isolating a 15 to 25 mersequence 5′ upstream from the hypermethylated CpG island for use as aMs-SNuPE primer. The present invention further provides a Ms-SNuPEprimer comprising a 15 to 25 mer oligonucleotide sequence obtained bythe process comprising, finding a hypermethylated CpG island in asomatic gene from a malignant tissue or cell culture, determining thesequence located immediately 5′ upstream from the hypermethylated CpGisland, and isolating a 15 to 25 mer sequence 5′ upstream from thehypermethylated CpG island for use as a Ms-SNuPE primer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the inventive Ms-SNuPE assay for determinationof strand-specific methylation status at cytosines. The process involvestreating genomic DNA with sodium bisulfite, and generating a template bya PCR technique for a top strand methylation analysis. Alternatively abottom strand methylation can also be assayed by designing theappropriate primers to generate a bottom strand-specific template. Theprocess further entails amplifying the templates by a PCR technique. ThePCR products are electrophoresed and isolated from agarose gels,followed by incubation with Ms-SNuPE primers, as disclosed hereinwherein the Ms-SNuPE primers comprise a from about a 15 mer to about a25 mer length primer that terminates immediately 5′ of a singlenucleotide to be assayed, and PCR buffer, [³²P]dNTPs and Taq polymerasefor primer extension reactions. The radiolabeled products are separated,for example, by electrophoresis on polyacrylamide gels under denaturingconditions and visualized by exposure to autoradiographic film orphosphorimage quantitation.

FIG. 2 shows the results from a quantitative methylation analysis ofthree top strand CpG sites from a 5′ CpG island of p16. P16 is a knowntumor suppressor gene and the particular region examined for changes inmethylation is the promoter region of this gene. The top panel providesthe locations of three sites analyzed (numbered 1, 2 and 3) relative tothe putative transcriptional start sites (vertical arrows pointingupwards) and the exon 1α coding domain. The PCR primers used for topstrand amplification of the 5′ region of p16 (which includes putativetranscriptional start sites) were 5′-GTA GGT GGG GAG GAG TTT AGT T-3′[SEQ ID NO. 13] and 5′-TCT AAT AAC CAA CCA ACC CCT CC-3′ [SEQ ID NO.14]. The control sets included “M” PCR product amplified from a plasmidcontaining bisulfide-specific methylated sequence; “U” PCR productamplified from a plasmid containing bisulfite-specific unmethylatedsequence; and “mix” a 50:50 mixture of methylated and unmethylatedPCR-amplified plasmid sequences. The DNA samples analyzed included T24and J82 bladder cancer cell lines; wbc (white blood cell), melanoma(primary melanoma tumor tissue sample), and bladder (primary bladdertumor tissue sample). The tissue samples were micro dissected fromparaffin-embedded tumor material. The grid at the bottom of the lowerpanel shows the ratio of methylated (C) versus unmethylated (T) bands ateach site based upon phosphorimage quantitation.

FIG. 3 shows a mixing experiment showing a linear response of theinventive Ms-SNuPE assay for detection of cytosine methylation. A T24bladder cancer cell line DNA (predominantly methylated) was added inincreasing amounts to a J82 bladder cancer cell line DNA (predominantlyunmethylated). FIG. 3 shows data from an 18 mer oligonucleotide [SEQ IDNO. 16] which was used in multiplex analysis of CpG methylation (site 2)of the p16 5′ CpG in combination with a 15-mer and 21-mer primer [SEQ IDNOS 17 and 15, respectively] (correlation coefficient=0.99). Both the 15mer and 21-mer produced a nearly identical linear response as the18-mer. FIG. 3 shows data from three separate experiments.

FIG. 4 shows a schematic diagram that outlines a process for ahigh-throughput methylation analysis. The Ms-SNuPE primer extensionreactions are performed and then the products are directly transferredto membranes, preferably nylon membranes. This allows for a large numberof samples to be analyzed simultaneously in a high-density format. Themembrane is washed and exposed to a phosphorimage cassette forquantitative methylation analysis and eliminate the need forpolyacrylamide gel electrophoresis for data measurement.

FIG. 5 (Panel A) shows results from quantitative analysis of DNAmethylation using the Ms-SNuPE blot transfer technique of FIG. 4. Levelsof DNA methylation in matched normal and tumor colon specimens wereanalyzed in the 5′ promoter region of the p16 gene. The averagemethylation of 3 sites in the p16 promoter (FIG. 2) was determined byquantitating the C:T signal ration by phosphorimage analysis. Panel Bshows the results of quantitating the average methylation of 3 CpG sitesusing standard polyacrylamide gel electrophoresis compared to dot blottransfers. The average methylation of the monitored sites in variouscolon specimens is plotted on the graph and shows little differencebetween quantitated values derived from polyacrylamide gelelectrophoresis compared tom the dotblot technique. These data show thefeasibility of using the Ms-SNuPE dotblot procedure for high-throughputdetection and quantitation of DNA methylation changes in cancer cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for determining DNA methylationpatterns at cytosine sites, comprising the steps of:

(a) obtaining genomic DNA from a DNA sample to be assayed, whereinsources of DNA include, for example, cell lines, blood, sputum, stool,urine, cerebrospinal fluid, paraffin-embedded tissues, histologicalslides and combinations thereof;

(b) reacting the genomic DNA with sodium bisulfite to convertunmethylated cytosine residues to uracil residues while leaving any5-methylcytosine residues unchanged to provide primers specific for thebisulfite-converted genomic sample for top strand or bottom strandmethylation analysis;

(c) performing a PCR amplification procedure using the top strand orbottom strand specific primers;

(d) isolating the PCR amplification products;

(e) performing a primer extension reaction using Ms-SNuPE primers,[³²P]dNTPs and Taq polymerase, wherein the Ms-SNuPE primers comprise afrom about a 15 mer to about a 22 mer length primer that terminatesimmediately 5′ of a single nucleotide to be assayed; and

(f) determining the relative amount of allelic expression of CpGmethylated sites by measuring the incorporation of different ³²P-labeleddNTPs.

Preferably, the [³²P]NTP for top strand analysis is [³²P]dCTP or[32P]TTP. Preferably, the [³²P]NTP for bottom strand analysis is[³²P]dATP or [1³²P]dGTP. Preferably, the isolation step of the PCRproducts uses an electrophoresis technique. Most preferably, theelectrophoresis technique uses an agarose gel.

DNA is isolated by standard techniques for isolating DNA from cellular,tissue or specimen samples. Such standard methods are found in textbookreferences such as Fritsch and Maniatis eds., Molecular Cloning: ALaboratory Manual, 1989.

The bisulfite reaction is performed according to standard techniques.For example and briefly, approximately 1 microgram of genomic DNA(amount of DNA can be less when using micro-dissected DNA specimens) isdenatured for 15 minutes at 45° C. with 2N NaOH followed by incubationwith 0.1 M hydroquinone and 3.6 M sodium bisulfite (pH 5.0) at 55° C.for 12 hours (appropriate range is 4-12 hours). The DNA is then purifiedfrom the reaction mixture using standard (commercially-available) DNAminiprep columns, or other standard techniques for DNA purification arealso appropriate. The purified DNA sample is resuspended in 55microliters of water and 5 microliters of 3N NaOH is added for adesulfonation reaction, preferably performed at 40° C. for 5-10 minutes.The DNA sample is then ethanol-precipitated and washed before beingresuspended in an appropriate volume of water. Bisulfite treatment ofDNA distinguishes methylated from unmethylated cytosines. The presentbisulfite treatment method has advantages because it is quantitative,does not use restriction enzymes, and many CpG sites can be analyzed ineach primer extension reaction by using a multiplex primer strategy.

The PCR amplification step (c) can be performed by standard PCRtechniques, following a manufacturer's instructions. For example,approximately 1-2 microliters of the bisulfite-treated DNA was used as atemplate for strand-specific PCR amplification in a region of interest.In a PCR reaction profile for amplifying a portion of the p16 5′ CpGisland, for example, a procedure of initial denaturation of 94° C. for 3minutes followed by a cycle of 94° C. of 30 seconds, 68° C. for 30seconds, 72° C. for 30 seconds for a total of 30 cycles. The PCRreactions were performed in 25 microliter volumes under conditionsof:˜50 ng bisulfite-converted DNA (less for micro dissected samples), 10mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl, 0.1% gelatin/ml, 100 μMof each of dNTP, 0.5 μM final concentration of each primer and 1 unit ofTaq polymerase. There are many chromatographic techniques that can beused to isolate the PCR amplification products. In one illustrativeprocedure, approximately 10-25 microliters of the amplified PCR productswere loaded onto 2% agarose gels and electrophoresed. The bands werevisualized and isolated using standard get purification procedures.

The primer extension reaction is conducted using standard PCR primerextension techniques but using Ms-SNuPE primers as provided herein.Approximately 10-50 nanograms of purified PCR template is used in eachMs-SNuPE reaction. A typical reaction volume is about 25 microliters andcomprises PCR template (about 10-50 ng), 1×PCR buffer, 1 μM of eachMs-SNuPE primer, 1 μCi of the appropriate ³²P-labeled dNTP (either[³²P]dCTP, [³²P]TTp, [³²P]dATP, [³²P]dGTP or combinations thereof), and1 unit of Taq polymerase. As a general rule, oligonucleotides used inthe primer extension reactions were designed to have annealingtemperatures within 2-3° C. of each other and did not hybridize tosequences that originally contained CpG dinucleotides. The Ms-SNuPEreactions were performed at 95° C. for 1 minute, 50° C. for 2 minutes,and 72° C. for 1 minute. A stop solution (10 microliters) was added tothe mixtures to terminate the reactions. The inventive Ms-SNuPE assayutilizes internal primer(s) which anneal to a PCR-generated template andterminate immediately 5′ of the single nucleotide to be assayed. Asimilar procedure has been used successfully for detection of genemutations Kuppuswamy et al., Proc. Natl. Acad. Sci. USA 88:1143-1147,1991) and for quantitation of allele-specific expression (Szabo andMann, Genes Dev. 9:3097-3108, 1995 and Greenwood and Burke, Genome Res.6:336-348, 1996).

There are several techniques that are able to determine the relativeamount of methylation at each CpG site, for example, using a denaturingpolyacrylamide gel to measure ³²P through phosphorimage analysis, ortransfer of Ms-SNuPE reaction products to nylon membranes, or even usingfluorescent probes instead of a ³²P marker. In one method fordetermining the relative amount of methylation at each CpG site,approximately 1-2 microliters of each Ms-SNuPE reaction product waselectrophoresed onto 15% denaturing polyacrylamide gel (7 M urea). Thegels were transferred to filter paper and then dried. Phosphorimageanalysis was performed to determine the relative amount of radiolabeledincorporation. An alternative method for determining the relative amountof methylation at individual CpG sites is by a direct transfer of theMs-SNuPE reaction products to nylon membranes. This technique can beused to quantitate an average percent methylation of multiple CpG siteswithout using polyacrylamide gel electrophoresis. High-throughputmethylation analysis was performed by direct transfer of the Ms-SNuPEreactions onto nylon membranes. A total of 100 microliters or 0.4 mMNaOH, 1 mM Na₄P₂O₇ was added to the completed primer extension reactionsinstead of adding stop solution. The mixture was directly transferred tonylon membranes using a dotblot vacuum manifold in a 96 well plateformat. Each vacuum transfer well was washed a total of 4 times with 200microliters of 2×SSC, 1 mM Na₄P₂O₇. The entire membrane was washed in2×SSC, 1 mM Na₄P₂O₇. The radioactivity of each spot on the dried nylonmembrane was quantitated by phosphorimaging analysis.

In the inventive quantitative Ms-SNuPE assay, the relative amount ofallelic expression is quantitated by measuring the incorporation ofdifferent ³²P-labeled dNTPs. FIG. 1 outlines how the assay can beutilized for quantitative methylation analysis. For example, the initialtreatment of genomic DNA with sodium bisulfite causes unmethylatedcytosine to be converted to uracil, which is subsequently replicated asthymine during PCR. Methylcytosine is resistant to deamination and isreplicated as cytosine during amplification. Quantitation of the ratioof methylated versus unmethylated cytosine (C versus T) at the originalCpG sites can be determined by incubating a gel-isolated PCR product,primer(s) and Taq polymerase with either [³²P]dCTP or [³²P]TTP, followedby denaturing polyacrylamide gel electrophoresis and phosphorimageanalysis. In addition, opposite strand (bottom strand) Ms-SNuPE primersare further designed which would incorporate either [³²P]dATP or[³²P]dGTP to assess methylation status depending on which CpG site isanalyzed.

Ms-SNuPE Primers

The present invention further provides a Ms-SNuPE primer sequencedesigned to anneal to and terminate immediately 5′ of a desired cytosinecodon in the CpG target site and that is located 5′ upstream from a CpGisland and are frequently hypermethylated in promoter regions of somaticgenes in malignant tissue. Preferably, the Ms-SNuPE primer sequencecomprises a sequence of at least fifteen bases having a sequenceselected from the group consisting of GaL1 (SEQ ID NO:1), GaL2 (SEQ IDNO:2), GaL4 (SEQ ID NQ:3), HuN1 (SEQ ID NO:4), HuN2 (SEQ ID NO:5), HuN3(SEQ ID NO:6), HuN4 (SEQ ID NO:7), HuN5 (SEQ ID NO:8), HuN6 (SEQ IDNO:9), CaS1 (SEQ ID NO:10), CaS2 (SEQ ID NO:11), CaS4 (SEQ ID NO:12),and combinations thereof. The present invention further provides amethod for obtaining a Ms-SNuPE primer sequence, comprising finding ahypermethylated CpG island in a somatic gene om a malignant tissue orcell culture, determining the sequence located immediately 5′ upstreamfrom the hypermethylated CpG island, and isolating a 15 to 25 mersequence 5′ upstream from the hypermethylated CpG island for use as aMs-SNuPE primer. The present invention further provides a Ms-SNuPEprimer comprising a 15 to 25 mer oligonucleotide sequence obtained bythe process comprising, (a) identifying hypermethylated CpG islands asomatic gene from a malignant tissue or cell culture source, (b)determining the sequence located immediately 5′ upstream from thehypermethylated CpG island, and (c) isolating at least a 15 mer sequence5′ upstream from the hypermethylated CpG island for use as a Ms-SNuPEprimer. Preferably the Ms-SNuPE primer sequence is from about 15 toabout 25 base pairs in length.

The ability to detect methylation changes associated with oncogenictransformation is of critical importance in understanding how DNAmethylation may contribute to tumorigenesis. Regions of DNA that havetumor-specific methylation alterations can be accomplished using avariety of techniques. This will permit rapid methylation analysis ofspecific CpG sites using the inventive quantitative Ms-SNuPE primerprocess. For example, techniques such as restriction landmark genomicscanning (RLGS) (Hatada et al., Proc. Natl. Acad. Sci. USA 88:9523-9527,1995), methylation-sensitive-representational difference analysis(MS-RDA) (Ushijima et al., Proc. Natl. Acad. Sci. USA 94:2284-2289,1997) and methylation-sensitive arbitrarily primed PCR (AP-PCR)(Gonzalgo et al., Cancer Res. 57: 594-599, 1997) can be used foridentifying and characterizing methylation differences between genomes.

Briefly, sequence determinations of regions of DNA that showtumor-specific methylation changes can be performed using standardtechniques, such as those procedures described in textbook referencessuch as Fritsch and Maniatis eds., Molecular Cloning: A LaboratoryManual, 1989. Additionally, commercially available kits or automated DNAsequencing systems can be utilized. Once specific regions of DNA havebeen identified by using such techniques, the Ms-SNuPE primers can beapplied for rapidly screening the most important CpG sites that areinvolved with the specific methylation changes associated with a cancerphenotype.

EXAMPLE 1

This example illustrates a quantitative methylation analysis of threetop strand sites in a 5′ CpG island of p16 in various DNA samples usingthe inventive method. The top panel provides the locations of threesites analyzed (numbered 1, 2 and 3) relative to the putativetranscriptional start sites (vertical arrows pointing upwards) and theexon 1α coding domain. The PCR primers used for top strand amplificationof the 5′ region of p16 (which includes putative transcriptional startsites) were 5′-GTA GGT GGG GAG GAG TTT AGT T-3′ [SEQ ID NO. 13] and5′-TCT AAT AAC CAACCA ACC CCT CC-3′ [SEQ ID NO. 14]. The reactions wereperformed in 25 μl total volume under the conditions of 50 ngbisulfite-treated DNA, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl,0.1% gelatin/ml, 100 μM of each dNTP, 0.5 μM final concentration of eachprimer and 1 U of Taq polymerase (Boehinger Mannheim, Indianapolis,Ind.). The reactions were hot-started using a 1:1 mixture ofTaq/TaqStart antibody (Clontech, Palo Alto, Calif.).

An initial denaturation of 94° C. for 3 minutes was followed by 94° C.for 30 sec, 68° C. for 30 sec, 72° C. for 30 sec for a total of 35cycles. The PCR products were separated by electrophoresis on 2% agarosegels and the bands were isolated using a Qiaquick™ gel extraction kit(Qiagen, Santa Clarita, Calif.).

The Ms-SNuPE reaction was performed in a 25 ml reaction volume with10-50 ng of PCR template incubated in a final concentration of 1×PCRbuffer, 1 μM of each Ms-SNuPE primer, 1 μCi of either [³²P]dCTP or[³²P]TTP and 1 U of Taq polymerase. The primer extensions were alsohot-started using a 1: mixture of Taq/TaqStart antibody. The primersused for the Ms-SNuPE analysis were: site 1 5′-TTT TTT TGT TTG GAA AGATAT-3′ [SEQ ID NO. 15]; site 2 5′-TTT TAG GGG TGT TAT ATT-3′ [SEQ ID NO.16]; site 3 5′-TTT GAG GGA TAG GGT-3′ [SEQ ID NO. 17]. The conditionsfor the primer extension reactions were 95° C. for 1 minute, 50° C. for2 minutes and 70° C. for 1 minute. A stop solution (10 μl) was added tothe reaction mixtures and the samples were loaded onto 15% denaturingpolyacrylamide gels (7 M urea). Radioactivity of the bands wasquantitated by phosphorimaging analysis. The control sets included “M”PCR product amplified from a plasmid containing bisulfide-specificmethylated sequence; “U” PCR product amplified from a plasmid containingbisulfite-specific unmethylated sequence; and “mix” a 50:50 mixture ofmethylated and unmethylated PCR-amplified plasmid sequences. The DNAsamples analyzed included T24 and J82 bladder cancer cell lines; wbc(white blood cell), melanoma (primary melanoma tumor tissue sample), andbladder (primary bladder tumor tissue sample). The tissue samples weremicro dissected from paraffin-embedded tumor material. The grid at thebottom of the lower panel shows the ratio of methylated (C) versusunmethylated (T) bands at each site based upon phosphorimagequantitation.

These data (FIG. 2) show the ability of the inventive assay to detectaltered patterns of methylation.

EXAMPLE 2

This example illustrates a mixing experiment showing a linear responseof the inventive Ms-SNuPE assay for detection of cytosine methylation. AT24 bladder cancer cell line DNA (predominantly methylated) was added inincreasing amounts to a J82 bladder cancer cell line DNA (predominantlyunmethylated). FIG. 3 shows data from an 18 mer oligonucleotide [SEQ IDNO. 16] which was used in multiplex analysis of CpG methylation (site 2)of the p16 5′ CpG in combination with a 15-mer and 21-mer primer [SEQ IDNOS 17 and 15, respectively] (correlation coefficient=0.99). Both the 15mer and 21-mer produced a nearly identical linear response as the18-mer. FIG. 3 shows data from three separate experiments. Differentialspecific activity and incorporation efficiency of each [³²P]dNTP wascontrolled for by using a 50:50 mixture of bisulfite-specific methylatedversus unmethylated PCR template for analysis.

EXAMPLE 3

This example provides a summary of DNA regions for which Ms-SNuPEprimers can be designed and the inventive method applied for aquantitative detection of abnormal DNA methylation in cancer cells. Thesequences are listed according to name, size and frequency ofhypermethylation in the corresponding cell line or primary tumor.

methylated in hypermethylated hypermethylated fragment size (bp) coloncell line in colon cancer in bladder cancer comments GaL1 530 {fraction(7/7)} (100%) {fraction (3/7)} (42%) {fraction (3/7)} (42%) GC content(0.6), observed/expected CpG (0.63) GaL2 308 {fraction (7/7)} (100%) ⅘(80%) {fraction (6/7)} (85%) GC content (0.6), observed/expected CpG(0.6) GaL4 177 {fraction (7/7)} (100%) ½ (50%) ¾ (75%) GC content(0.59), observed/expected CpG (0.50) CaS1 215 {fraction (4/7)} (57%) {fraction (0/5)} (0%)  {fraction (2/7)} (28%) GC content (0.55),observed/expected CpG (0.78) CaS2 220 {fraction (4/7)} (57%)  ⅗ (60%){fraction (3/7)} (42%) GC content (0.54), observed/expected CpG (0.74)CaS4 196 {fraction (6/7)} (85%)  {fraction (0/5)} (0%)  {fraction (1/7)}(14%) GC content (0.64), observed/expected CpG (0.84) HuN1 148 {fraction(7/7)} (100%) ⅗ (60%) {fraction (3/7)} (42%) GC content (0.54),observed/expected CpG (0.99) HuN2 384 {fraction (7/7)} (100%) ⅘ (80%){fraction (2/7)} (28%) GC content (0.6), observed/expected CpG (0.62)HuN3 178 {fraction (6/7)} (85%)  ⅘ (80%) {fraction (3/7)} (42%) GCcontent (0.53), observed/expected CpG (0.97) HuN4 359 {fraction (7/7)}(100%) ⅗ (60%) {fraction (4/7)} (57%) GC content (0.51),observed/expected CpG (0.47) HuN5 251 {fraction (7/7)} (100%) ⅖ (40%){fraction (5/7)} (71%) GC content (0.63), observed/expected CpG (0.77)HuN6 145 {fraction (6/7)} (85%)  ¾ (75%) ½ (50%) GC content (0.55),observed/expected CpG (0.47)

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 17 <210> SEQ ID NO 1 <211> LENGTH: 530<212> TYPE: DNA <213> ORGANISM: homo sapiens <400> SEQUENCE: 1cccgcgacct aagccagcga cttaccacgt tagtcagcta agaagtggca ga#gctgggat     60tcgaacctat aaagaactct gaagcctggg tatttttaca tgacacttta ca#taatgcgc    120cacggggtag tcggaggggg aggtccatct ccctttccct tgctgtccat ct#ccacagaa    180aagaagcaag tggaggacag gagccagaaa gtcatctggc cgcggatcat tc#cggagtga    240cccccgccgc caccactcgc atagtccgct tatggcggga gggcacctca ga#gattctca    300caggggctgt gcggccagaa ccagaagtgc aaagcaccgt tagcgactct at#cgccccct    360gccgcctgtg gcgcccagtc cgaagctgct gttttcagga gggctagtgg gc#taagaaaa    420gagctcaccg actgactgcc caacagctgt tgcgagccag tgctaggctg ca#gacagcct    480tgccaaatgt ggtgacataa gcgggagggg ggaacattta gagagcccta  #             530 <210> SEQ ID NO 2 <211> LENGTH: 308 <212> TYPE: DNA<213> ORGANISM: homo sapiens <400> SEQUENCE: 2ctagggtagg ctggtctgtg ctggatacgc gtgttcttct gcggagttaa ag#ggtcgggg     60acgggggttc tggacttacc agagcaattc cagccggtgg gcgtttgaca gc#cacttaag    120gaggtaggga aagcgagctt caccgggcgg gctacgatga gtagcatgac gg#gcagcagc    180agcagcagcc agcaaaagcc tagcaaagtg tccagctgct gcactgccgc gg#ggactccc    240acatcaccat gactagttgt gcaactctgc agcagaaacg gcttccgagg aa#cacaggat    300 cgcggggg                 #                  #                   #         308 <210> SEQ ID NO 3 <211> LENGTH: 177<212> TYPE: DNA <213> ORGANISM: homo sapiens <400> SEQUENCE: 3gcttcctttt tctcggcttt cctcactatc ctctccctgt tcgagagtat ct#ccaccagc     60accgagcctc acacgggctg tgcctccatc tttggaatgc ctacccttct tt#cttgcgaa    120gcccctccca gggccagccc ttgtgcaccg gctcaagggg actgctctcc tg#cctcg       177 <210> SEQ ID NO 4 <211> LENGTH: 148 <212> TYPE: DNA<213> ORGANISM: homo sapiens <400> SEQUENCE: 4ttgcgccgat cgtcaagaac ctctcatccc tggcagcagc aaagccaata ta#tttccatt     60tcttatttca gtttgccacc aaaacaaagc tgcgcgcggc tgagggcagg aa#ggcgctga    120 gaccgaccga gaagaaggga cgtcccgg         #                   #            148 <210> SEQ ID NO 5 <211> LENGTH: 384<212> TYPE: DNA <213> ORGANISM: homo sapiens <400> SEQUENCE: 5caggcccgcc gagactccac tccaactacc aggaaatttc ccgtggagct tc#aattcctg     60ggaccctcct actgcgggga gagtggtttc cctgccccac accatgccct ag#gcccgagt    120ctgcggctct tgggggatct ctccgagctc cgacaccgtg ttcggaccgg gt#gcgccctg    180ccgctggggc tcaagcctgc aggcgtgaga accgggggac tctctatggc ac#caagagct    240tcaccgtgag cgtaggcaga agcttcgctt tgatcctagg gcttacaaag tc#ctcctttg    300gctgcccatg atggtaaaag ggcagttgct cacaaagcgc gagtgtgtgt gc#cagacagt    360 gtaaatgagt gttgggaccg gcgt          #                   #               384 <210> SEQ ID NO 6<211> LENGTH: 178 <212> TYPE: DNA <213> ORGANISM: homo sapiens<400> SEQUENCE: 6gggtccgttc gtgaatgcat gagcagggtg tgagcgccag ggggttacac tt#ctcacggg     60ttaaaaccca gacaacttca cgagggaacc acgtgccatt ttaacagcgt ac#ggtcggga    120tcgtgggacg tcattaaacg gagtgggttg agtatgtgac tctgtcaccc at#tttctg      178 <210> SEQ ID NO 7 <211> LENGTH: 359 <212> TYPE: DNA<213> ORGANISM: homo sapiens <400> SEQUENCE: 7ccccgcgggg cagaatccaa gtgagtcaga cacattgctc cctccctgct gc#tgccagtc     60catctctttg ccaacaaacc tgcttaaaat gccaaagctg gtccaaagtt tc#aggaaaac    120aacttccgcc agagggcacg tagagggcac agatgctata gatgcttctc tg#acaaacac    180tcctgacccc cttgacagat tggaaaatac atggttcaga aagggtgaga ga#tttcaact    240tgagaagtga aactaggaaa agatggaagg tgtccggatt tctagctcaa gt#ccacacac    300tgcttctgct gcggtgacta aatcgtggct gtgttctcat cacctgcctc gc#ggcgcgc     359 <210> SEQ ID NO 8 <211> LENGTH: 251 <212> TYPE: DNA<213> ORGANISM: homo sapiens <400> SEQUENCE: 8ggcgggcctg ggcaccgcgg agggggggct tttctgcgcc cggcgaagcg tg#gaacttgc     60gccctgaggc agcgcggcga gaccagtcca gagaccgggg cgagcctcct ca#ggattcct    120cgccccagtg cagatgctgt gagcttagac gaggacaggg catggcactc gg#cttggccc    180gtagtggacg gtgtttttgc agtcatgaac ccaaacgccg caaaccttga cc#gtttcccc    240 acccgtgttg t                #                  #                   #      251 <210> SEQ ID NO 9 <211> LENGTH: 145<212> TYPE: DNA <213> ORGANISM: homo sapiens <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (126)..(126)<223> OTHER INFORMATION: undetermined base residue <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (127)..(127)<223> OTHER INFORMATION: undetermined base residue <400> SEQUENCE: 9tgagagcagc atcctcccct gcgtgtggtt ctctaactta cctcctgtat gg#ggtctgcg     60gacccagcac acctcccggg cccccaaaaa attccagctc aagagcccta aa#aatcctta    120 ccctgnnaaa gtttgagctt ctccc          #                   #              145 <210> SEQ ID NO 10<211> LENGTH: 215 <212> TYPE: DNA <213> ORGANISM: homo sapiens<400> SEQUENCE: 10acgccggcca cagttcttca gtgaaacgct tcactctctg gtcatagagg ta#ggaaacta     60tagctgtccc aactaaatgt caggacgaat tagcccagct ggtcacgctc ac#agtcaccg    120cctccaccag actgagcgac cctcccaacg gggtttgccg tgttgggagg ac#agcggagt    180 ttcgttgctg tgtcaatttg tgtagacgcg gctgc       #                   #      215 <210> SEQ ID NO 11 <211> LENGTH: 220<212> TYPE: DNA <213> ORGANISM: homo sapiens <400> SEQUENCE: 11ctgctctctt ctcttctttt cccctttcct ctcctctccc tttcctcagg tc#acagcgga     60gtgaatcagc tcggtggtgt ctttgtcaac gggcggccac tgccggactc ca#cccggcag    120aagattgtag agctagctca cagcggggcc cggccgtgcg acatttcccg aa#ttctgcag    180 gtgatcctcc cggcgccgcc ccactcgccg cccccgcggc     #                   #   220 <210> SEQ ID NO 12 <211> LENGTH: 196<212> TYPE: DNA <213> ORGANISM: homo sapiens <400> SEQUENCE: 12gggcggcacg gagggagtca ggagtgagcc cgaagatgga gagaagtcga tt#cgcccaga     60gaacgcaaga cggtggatca gagatgagtc ccaggaacct cagagagcga gg#ctgacagg    120cccggggaga ggaccgggca gggacaaacc agcggacaga gcagagcgcg aa#atggttga    180 gaccgggaag cgacct              #                  #                   #   196 <210> SEQ ID NO 13 <211> LENGTH: 22<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: p16 promoter region-specific # Ms-SNuPE primer<400> SEQUENCE: 13 gtaggtgggg aggagtttag tt           #                   #                 22 <210> SEQ ID NO 14<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: p16 promoter region-specific# Ms-SNuPE primer <400> SEQUENCE: 14tctaataacc aaccaacccc tcc            #                  #                23 <210> SEQ ID NO 15 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: p16 promoter region-specific # Ms-SNuPE primer<400> SEQUENCE: 15 tttttttgtt tggaaagata t           #                   #                   #21 <210> SEQ ID NO 16<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: p16 promoter region-specific# Ms-SNuPE primer <400> SEQUENCE: 16 ttttaggggt gttatatt             #                   #                   #  18 <210> SEQ ID NO 17<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: p16 promoter region-specific# Ms-SNuPE primer <400> SEQUENCE: 17 tttgagggat agggt              #                   #                   #    15

We claim:
 1. A diagnostic method for colon or bladder cancer,comprising: (a) obtaining an isolated test genomic DNA sample from atest tissue; (b) subjecting the test genomic DNA sample to methylationanalysis, whereby the methylation state of one or more CpG dinucleotidesequences within a sequence selected from the group consisting of SEQ IDNOS:1-9 and 11, and fragments thereof comprisin at least 16 contiguousbases is determined; and (c) comparing the methylation state of the oneor more CpG dinucleotide sequences of the test sample with that ofcorresponding sequences of a reference genomic sample from a referencegenomic tissue, or with that of a corresponding known referencemethylation state, thereby providing, at least in part, a colon orbladder cancer diagnosis.
 2. The diagnostic method of claim 1, whereinthe test tissue is cancer tissue or putative cancer tissue, and whereinthe reference tissue or reference methylation state is that ofcorresponding normal tissue, or corresponds to a known methylation stateof corresponding normal tissue, respectively.
 3. The diagnostic methodof claim 1, wherein the methylation analysis comprises a method selectedfrom the group consisting of DNA sequencing using bisulfite treatment,restriction landmark genomic scanning, methylation-sensistie arbitrarilyprimed PCR, Southern analysis using a methylation-sensitive restrictionenzyme, methylation-specific PCR, restriction enzyme digestion of PCRproducts amplified from bisulfite-converted DNA, and combinationsthereof.
 4. The diagnostic method of claim 1, where the methylationanalysis comprises: (a) reacting the test genomic DNA sample with sodiumbisulfite to convert unmethylated cytosine residues to uracil residueswhile leaving any 5-methylcytosine residues unchanged to create anexposed bisulfite-converted DNA sample having binding sites for primersspecific for the bisulfite-converted DNA sample; (b) performing a PCRamplification procedure using top strand or bottom strand specificprimers; (c) isolating the PCR amplification products; (d) performing aprimer extension reaction using a Ms-SNuPE primer, dNTPs and Taqpolymerase, wherein the Ms-SNuPE primer comprises from about a 15-mer toabout a 22-mer length primer sequence that is complementary to thebisulfite-converte DNA sample and terminates immediately 5′ of thecytosine residue of the one or more CpG dinucleotide sequences to beassayed; and (f) determining the methylation state of the one or moreCpG dinucleotide sequences by determining the identity of the firstprimer-extended base.
 5. The diagnostic method of claim 4, wherein thedNTPs are labeled with a label selected from the group consisting ofradiolabels and fluorescent labels, and wherein determining the identityof the first primer-extended base is by measuring incorporation of thelabeled dNTPs.
 6. A diagnostic method for bladder cancer, comprising:(a) obtaining an isolated test genomic DNA sample from a test tissue;(b) subjecting the test genomic DNA sample to methylation analysis,whereby the methylation state of one or more CpG dinucleotide sequenceswithin a sequence selected from the group consisting of SEQ ID NOS:1-9and 11, and fragments thereof comprising at least 16 contiguous bases isdetermined; and (c) comparing the methylation state of the one or moreCpG dinucleotide sequences of the test sample with that of correspondingsequences of a reference genomic sample from a reference genomic tissue,or with that of a corresponding known reference methylation state,thereby providing, at least in part, a bladder cancer diagnosis.
 7. Thediagnostic method of claim 6, wherein the test tissue is cancer tissueor putative cancer tissue, and wherein the reference tissue or referencemethylation state is that of corresponding normal tissue, or correspondsto a known methylation state of corresponding normal tissue,respectively.
 8. The diagnostic method of claim 6, wherein themethylation analysis comprises a method selected from the groupconsisting of DNA sequencing using bisulfite treatment, restrictionlandmark genomic scanning, methylation-sensistive arbitrarily primedPCR, Southern analysis using a methylation-sensitive restriction enzyme,methylation-specific PCR, restriction enzyme digestion of PCR productsamplified from bisulfite-converted DNA, and combinations thereof.
 9. Thediagnostic method of claim 6, wherein the methylation analysiscomprises: (a) reacting the test genomic DNA sample with sodiumbisulfite to convert unmethylated cytosine residues to uracil residueswhile leaving any 5-methylcytosine residues unchanged to create anexposed bisulfite-converted DNA sample having binding sites for primersspecific for the bisulfite-converted DNA sample; (b) performing a PCRamplification procedure using top strand or bottom strand specificprimers; (c) isolating the PCR amplification products; (d) performing aprimer extension reaction using a Ms-SNuPE primer, dNTPs and Taqpolymerase, wherein the Ms-SNuPE primer comprises from about a 15-mer toabout a 22-mer length primer sequence that is complementary to thebisulfite-converte DNA sample and terminates immediately 5′ of thecytosine residue of the one or more CpG dinucleotide sequences to beassayed; and (f) determining the methylation state of the one or moreCpG dinucleotide sequences by determining the identity of the firstprimer-extended base.
 10. The diagnostic method of claim 9, where thedNTPs are labeled with a label selected from the group consisting ofradiolabels and fluorescent labels, and wherein determining the identityof the first primer-extended base is by measuring incorporation of thelabeled dNTPs.